The sequence polymorphisms of intergenic transcribed spacer and the antifungal susceptibility profile of 18 Trichosporon asahii isolates from Spain, Argentina, and Brazil together with 43 intergenic transcribed spacer 1 sequences deposited in the GenBank were analyzed. Six genotypes were detected instead of 5 genotypes described previously. Genotype 1 was the most common found comprising 57.3% of all strains, followed by genotype 3 (14.7%) and genotype 5 (13.1%). Spanish strains had members in all genotypes except 2, whereas South American isolates were grouped with genotypes 1, 3, and 6. Our results indicate that all genotypes are present in at least 2 countries suggesting a worldwide distribution. On the other hand, genotype 6 was not previously described but was only composed of 2 South American strains isolated from a subcutaneous abscess and skin. All isolates showed amphotericin B MICs>or=2 mg/L supporting the in vitro resistance of this species to this antifungal. Three isolates from South America showed high MICs to all antifungals analyzed. The true epidemiologic usefulness of classifying T. asahii in genotypes should be ascertaining analyzing a high number of isolates from many countries.